Selective Enrichment of Nitrososphaera viennensis-Like Ammonia-Oxidizing Archaea over Ammonia-Oxidizing Bacteria from Drinking Water Biofilms

ABSTRACT Ammonia-oxidizing archaea (AOA) can oxidize ammonia to nitrite for energy gain. They have been detected in chloraminated drinking water distribution systems (DWDS) along with the more common ammonia-oxidizing bacteria (AOB) and nitrite-oxidizing bacteria (NOB). To date, no members of the AOA have been isolated or enriched from drinking water environments. To begin the investigation of the role of AOA in chloraminated DWDS, we developed a selective approach using biofilm samples from a full-scale operational network as inoculum. A Nitrososphaera viennensis-like AOA taxon was enriched from a mixed community that also included Nitrosomonas-like AOB while gradually scaling up the culture volume. Dimethylthiourea (DMTU) and pyruvate at 100 μM were added to promote the growth of AOA while inhibiting AOB. This resulted in the eventual washout of AOB, while NOB were absent after 2 or 3 rounds of amendment with 24 μM sodium azide. The relative abundance of AOA in the enrichment increased from 0.2% to 39.5% after adding DMTU and pyruvate, and further to 51.6% after filtration through a 0.45-μm pore size membrane, within a period of approximately 6 months. IMPORTANCE Chloramination has been known to increase the risk of nitrification episodes in DWDS due to the presence of ammonia-oxidizing microorganisms. Among them, AOB are more frequently detected than AOA. All publicly available cultures of AOA have been isolated from soil, marine or surface water environments, meaning they are allochthonous to DWDS. Hence, monochloramine exposure studies involving these strains may not accurately reflect their role in DWDS. The described method allows for the rapid enrichment of autochthonous AOA from drinking water nitrifying communities. The high relative abundance of AOA in the resulting enrichment culture reduces any confounding effects of co-existing heterotrophic bacteria when investigating the response of AOA to varied levels of monochloramine in drinking water.

. Maximum likelihood phylogenetic tree based on archaeal 16S rRNA gene nucleotide sequences. The cloned archaeal 16S rRNA sequences are most related to the 16S rRNA gene sequence from Nitrososphaera viennensis strain EN76 (closed circle). The evolutionary history was inferred by using the Maximum Likelihood method and Tamura-Nei model (80). The unrooted tree with the highest log likelihood (-8752.35) is shown. Initial tree(s) for the heuristic search were obtained automatically by applying Neighbor-Join and BioNJ algorithms to a matrix of pairwise distances estimated using the Maximum Composite Likelihood (MCL) approach, and then selecting the topology with superior log likelihood value. A discrete Gamma distribution was used to model evolutionary rate differences among sites (5 categories (+G, parameter = 1.0804)). The tree is drawn to scale, with branch lengths measured in the number of substitutions per site. This analysis involved 34 nucleotide sequences. All positions with less than 90% site coverage were eliminated, i.e., fewer than 10% alignment gaps, missing data, and ambiguous bases were allowed at any position (partial deletion option). There was a total of 1397 positions in the final dataset. The number of bootstrap replications was 500. Evolutionary analyses were conducted in MEGA X (67, 81). Figure S2. Maximum likelihood phylogenetic tree based on archaeal amoA gene nucleotide sequences. The cloned archaeal amoA sequences (closed triangles) are most closely related to the amoA sequence from Nitrososphaera viennensis strain EN76 (closed circle). The evolutionary history was inferred by using the Maximum Likelihood method and General Time Reversible model (80). The unrooted tree with the highest log likelihood (-4008.34) is shown. The percentage of trees in which the associated taxa clustered together is shown next to the branches. Initial tree(s) for the heuristic search were obtained automatically by applying Neighbor-Join and BioNJ algorithms to a matrix of pairwise distances estimated using the Maximum Composite Likelihood (MCL) approach, and then selecting the topology with superior log likelihood value. A discrete Gamma distribution was used to model evolutionary rate differences among sites (5 categories (+G, parameter = 1.0704)). The rate variation model allowed for some sites to be evolutionarily invariable ([+I], 37.69% sites). The tree is drawn to scale, with branch lengths measured in the number of substitutions per site. This analysis involved 30 nucleotide sequences. Codon positions included were 1st+2nd+3rd+Noncoding. All positions with less than 90% site coverage were eliminated, i.e., fewer than 10% alignment gaps, missing data, and ambiguous bases were allowed at any position (partial deletion option). There was a total of 600 positions in the final dataset. The number of bootstrap replications was 500. Evolutionary analyses were conducted in MEGA X (67, 81). Figure S3. Maximum likelihood phylogenetic tree based on translated peptide sequences of cloned archaeal amoA nucleotide sequences. The translated peptide sequences of the cloned archaeal amoA are closely related to the amoA peptide sequences from the AOA genera Nitrososphaera, including the species Nitrososphaera viennensis (closed circle). The cloned sequences are highlighted by closed triangles. The evolutionary history were inferred using the Maximum Likelihood method (80) and Le_Gascuel_2008 model (82). The unrooted tree with the highest log likelihood (-1321.91) is shown. Initial tree(s) for the heuristic search were obtained automatically by applying Neighbor-Join and BioNJ algorithms to a matrix of pairwise distances estimated using a JTT model, and then selecting the topology with superior log likelihood value. A discrete Gamma distribution was used to model evolutionary rate differences among sites (5 categories (+G, parameter = 0.2277)). The tree is drawn to scale, with branch lengths measured in the number of substitutions per site. This analysis involved 36 amino acid sequences. All positions with less than 90% site coverage were eliminated, i.e., fewer than 10% alignment gaps, missing data, and ambiguous bases were allowed at any position (partial deletion option). The final dataset had a total of 210 positions. The number of bootstrap replications was 500. Evolutionary analyses were conducted in MEGA X (67, 81). Figure S4. Maximum likelihood phylogenetic tree based on 16S amplicon sequence variant (ASV) nucleotide sequences, which are closely related to the 16S sequences from the Nitrosomonas oligotropha group (closed circles) of AOB. The ASV sequences are highlighted by the filled diamonds. The evolutionary history was inferred by using the Maximum Likelihood method and Kimura 2-parameter model (80). The unrooted tree with the highest log likelihood (-883.41) is shown.
The percentage of trees in which the associated taxa clustered together is shown next to the branches. Initial tree(s) for the heuristic search were obtained automatically by applying Neighbor-Join and BioNJ algorithms to a matrix of pairwise distances estimated using the Maximum Composite Likelihood (MCL) approach, and then selecting the topology with superior log likelihood value. A discrete Gamma distribution was used to model evolutionary rate differences among sites (5 categories (+G, parameter = 0.7320)). The rate variation model allowed for some sites to be evolutionarily invariable ([+I], 43.93% sites). The tree is drawn to scale, with branch lengths measured in the number of substitutions per site. This analysis involved 27 nucleotide sequences.
All positions with less than 90% site coverage were eliminated, i.e., fewer than 10% alignment gaps, missing data, and ambiguous bases were allowed at any position (partial deletion option). There were a total of 321 positions in the final dataset. The number of bootstrap replications was 500.
Evolutionary analyses were conducted in MEGA X (67, 81). Figure S5. Maximum likelihood phylogenetic tree based on the cloned bacterial amoA nucleotide sequences, which are diversely related to amoA nucleotide sequences from the AOB genera Nitrosomonas and Nitrosococcus, including the species Nitrosomonas oligotropha (closed circle).
The cloned sequences are highlighted by the filled diamonds. The evolutionary history was inferred by using the Maximum Likelihood method and Kimura 2-parameter model (80). The unrooted tree with the highest log likelihood (-4809.00) is shown. The percentage of trees in which the associated taxa clustered together is shown next to the branches. Initial tree(s) for the heuristic search were obtained automatically by applying Neighbor-Join and BioNJ algorithms to a matrix of pairwise distances estimated using the Maximum Composite Likelihood (MCL) approach, and then selecting the topology with superior log likelihood value. A discrete Gamma distribution was used to model evolutionary rate differences among sites (5 categories (+G, parameter = 1.0460)). The rate variation model allowed for some sites to be evolutionarily invariable ([+I], 25.57% sites). The tree is drawn to scale, with branch lengths measured in the number of substitutions per site. This analysis involved 28 nucleotide sequences. All positions with less than 90% site coverage were eliminated, i.e., fewer than 10% alignment gaps, missing data, and ambiguous bases were allowed at any position (partial deletion option). There were a total of 479 positions in the final dataset. The number of bootstrap replications was 500. Evolutionary analyses were conducted in MEGA X (67, 81). including Nitrosomonas oligotropha (closed circle). The cloned sequences are highlighted by closed diamonds. One of the cloned sequences is diversely related to the amoA peptide sequences from the AOB genera Nitrosomonas, Nitrosospira and Nitrosococcus. The evolutionary history was inferred using the Maximum Likelihood method (80) and Le_Gascuel_2008 model (82). The unrooted tree with the highest log likelihood (-1454.32) is shown. Initial tree(s) for the heuristic search were obtained automatically by applying Neighbor-Join and BioNJ algorithms to a matrix of pairwise distances estimated using a JTT model, and then selecting the topology with superior log likelihood value. A discrete Gamma distribution was used to model evolutionary rate differences among sites (5 categories (+G, parameter = 0.7441)). The tree is drawn to scale, with branch lengths measured in the number of substitutions per site. This analysis involved 33 amino acid sequences.
All positions with less than 90% site coverage were eliminated, i.e., fewer than 10% alignment gaps, missing data, and ambiguous bases were allowed at any position (partial deletion option). There was a total of 144 positions in the final dataset. The number of bootstrap replications was 500.
Evolutionary analyses were conducted in MEGA X (67, 81). A B Figure S7. Comparison of ddPCR reactions using NSS_amoAF/R and Arch_amoAF/R primers. 1D plots from the archaeal amoA ddPCR droplet reads showed good signal separation for the positive droplets from the negative droplets of the reactions using the designed primers (A), but the signal separation between the positive and negative droplets of the reactions using the published primers (71) was poorer (B).  H10  H09  H08  G10  G09  G08  F10  F09  F08  E10  E09  E08  D10  D08  C10  C09  C08 12500 A08 B08 B09B10 Figure S9. The total taxa sequenced from the enrichment without treatment. The taxa are faceted by genera and colored by phyla. Proteobacteria phylum appeared to be the dominant taxa, with Hyphomicrobium being the dominant genus in the enrichment. NA refers to taxa not identifiable to genus level.